Genetics: From Genes to Genomes PowerPoint to accompany Genetics: From Genes to Genomes Fourth Edition Leland H. Hartwell, Leroy Hood, Michael L. Goldberg, Ann E. Reynolds, and Lee M. Silver Prepared by Mary A. Bedell University of Georgia Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

8 Gene Expression: The Flow of Information from DNA to RNA to Protein PART II What Genes Are and What They Do CHAPTER CHAPTER Gene Expression: The Flow of Information from DNA to RNA to Protein CHAPTER OUTLINE 8.1 The Genetic Code 8.2 Transcription: From DNA to RNA 8.3 Translation: From mRNA to Protein 8.4 Differences in Gene Expression Between Prokaryotes and Eukaryotes 8.5 A Comprehensive Example: Computerized Analysis of Gene Expression in C. elegans 8.6 The Effect of Mutations on Gene Expression and Gene Function Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8

Four general themes for gene expression Pairing of complementary bases is the key to the transfer of information from DNA to RNA and from RNA to protein Polarities of DNA, RNA, and polypeptides help guide the mechanisms of gene expression Gene expression requires input of energy and participation of specific proteins and macromolecular assemblies Mutations that change genetic information or obstruct the flow of its expression can have dramatic effects on phenotype Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8

Gene expression: the flow of genetic information from DNA via RNA to protein RNA polymerase transcribes DNA to produce an RNA transcript Ribosomes translate the mRNA sequence to synthesize a polypeptide Translation follows the "genetic code" Fig. 8.2 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8

Triplet codons of nucleotides represent individual amino acids 61 codons represent the 20 amino acids 3 codons signify stop Fig. 8.3 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8

A gene's nucleotide sequence is colinear with the amino acid sequence of the encoded polypeptide Charles Yanofsky – deduced key features of relationship between nucleotides and amino acids Generated large number of trp− auxotrophic mutants in E. coli Detailed analysis of mutations in trpA gene TrpA encodes a subunit of tryptophan synthetase Fine structure genetic map of trpA gene based on intragenic recombination Determined amino acid sequences of mutant tryptophan synthetase Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8

Mutations in a gene are colinear with the sequence of amino acids in the encoded polypeptide Fig. 8.4a Different point mutations may affect the same amino acid Codons must contain >1 nucleotide Each point mutation affects only one amino acid Each nucleotide is part of only one codon Fixed no third p in polypeptide Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8

Evidence that codons must contain two or more base pairs Intragenic recombination Wild-type allele can be produced by crossing two mutant strains with different amino acids at the same position Fig. 8.4b Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8

Studies of frameshift mutations showed that codons consist of three nucleotides F. Crick and S. Brenner (1955) Proflavin-induced mutations in bacteriophage T4 rIIB gene Intercalates into DNA Causes insertions and deletions 2nd treatment with proflavin can create a 2nd mutation that restores wild-type function (revertant) Intragenic suppression) Fig. 8.5 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8

Different sets of T4 rIIB mutations generate either a mutant or a normal phenotype Codons must be read in order from a fixed starting point Starting point establishes a reading frame Intragenic supression occurs only when wild-type reading frame is restored Fig. 8.5d Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8

Codons consist of three nucleotides read in a defined reading frame Counterbalancing of mutations (e.g. +1 and -1) can restore the reading frame Intragenic suppression occurs when mutations involve multiples of three Fig. 8.6 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8

Codons consist of three nucleotides read in a defined reading frame (cont) Frameshift mutations alter the reading frame of codons after the point of insertion or deletion Fig. 8.6 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8

Cracking the code: Which codons represent which amino acids? Several technological breakthroughs in 1950s and 1960s Discovery of mRNA In vitro translation of synthetic mRNAs Preparation of cellular extracts that allowed translation in a test tube Developed techniques to synthesize artificial RNAs with known nucleotide sequence Allowed synthesis of simple polypeptides Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8

Cracking the code: Discovery of mRNA 1950s, studies in eukaryotic cells Evidence that protein synthesis takes place in cytoplasm Deduced from radioactive tagging of amino acids Implies that there must be a molecular intermediate between genes in the nucleus and protein synthesis in the cytoplasm Discovery of messenger RNAs (mRNAs), molecules for transporting genetic information Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8

Using synthetic mRNAs and in vitro translation to crack the genetic code 1961 – Marshall Nirenberg and Heinrich Mathaei Added artificial mRNAs to cell-free translation systems Fig. 8.7a Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8

The coding possibilities of synthetic mRNAs Polypeptides synthesized Simple polypeptides are encoded by simple polynucleotides Fig. 8.7b Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8

Cracking the genetic code with mini-mRNAs Nirenberg and Leder (1965) Resolved ambiguities in genetic code In vitro translation with trinucleotide synthetic mRNAs and tRNAs charged with a radioactive amino acid Fig. 8.8 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8

Correlation of polarities in DNA, mRNA, and polypeptide Template strand of DNA is complementary to mRNA and to the RNA-like strand of DNA 5’-to-3’ in the mRNA corresponds to N-to-C-terminus in the polypeptide Fig. 8.9 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8

Summary of the genetic code Genetic code has triplet codons Codons are nonoverlapping Three nonsense codons don’t encode an amino acid; UAA (ocher), UAG (amber) and UGA (opal) Genetic code is degenerate Reading frame is established from a fixed starting point – codon for translation initiation is AUG mRNAs and polypeptides have corresponding polarities Mutations can be created in three ways; frameshift, missense, and nonsense Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8

Experimental verification of the genetic code Yanofsky: Single-base substitutions can explain the altered amino acids in trp− and trp+ revertants Position in polypeptide Amino acid in wild-type polypeptide Missense mutations are single nucleotide substitutions and conform to the code Amino acid in mutant polypeptide Fig. 8.10a Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8

Experimental verification of the genetic code (cont) Yanofsky: Amino acid alterations that explain intragenic suppression of proflavin-induced frame-shift mutations Fig. 8.10b Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8

Genetic code is almost, but not quite, universal Virtually all cells alive now use the same basic genetic code In vitro translational systems from one organism can use mRNA from another organism to generate protein Comparisons of DNA and protein sequence reveal perfect correspondence between codons and amino acids among all organisms Genetic code must have evolved early in history of life Exceptional genetic codes found in ciliates and mitochondria Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8

Transcription: From DNA to RNA RNA polymerase catalyzes transcription Promoters are DNA sequences that provide the signal to RNA polymerase for starting transcription RNA polymerase adds nucleotides in 5’-to-3’ direction Formation of phosphodiester bonds using ribonucleotide triphosphates (ATP, CTP, GTP, and UTP) Hydrolysis of bonds in NTPs provides energy for transcription Terminators are RNA sequences that provide the signal to RNA polymerase for stopping transcription Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8

Transcription in bacterial cells Initiation: The beginning of transcription RNA polymerase binds to promoter sequence located near beginning of gene Sigma (s) factor binds to RNA polymerase ( holoenzyme) Region of DNA is unwound to form open promoter complex Phosphodiester bonds formed between first two nucleotides Fig. 8.11a Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8

Transcription in bacterial cells Elongation: An RNA copy of the gene factor separates from RNA polymerase ( core enzyme) Core RNA polymerase loses affinity for promoter, moves in 3’-to-5’ direction on template strand Within transcription bubble, NTPs added to 3’ end of nascent mRNA Fig. 8.11b Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8

Transcription in bacterial cells Termination: The end of transcription Terminators are RNA sequences that signal the end of transcription Two kinds of terminators in bacteria: extrinsic (require rho factor) and intrinsic (don’t require additional factors) Usually form hairpin loops (intramolecular H-bonding) Fig. 8.11c Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8

The product of transcription is a single-stranded primary transcript Fig. 8.11d Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8

The promoters of 10 different bacterial genes Most promoters are upstream to the transcription start point RNA polymerase makes strong contacts at -10 and -35 Fig. 8.12 Strong E. coli promoters Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8

Structure of the methylated cap at the 5' end of eukaryotic mRNAs Capping enzyme adds a "backward" G to the 1st nucleotide of a primary transcript Transcribed bases Methylated cap – not transcribed Triphosphate bridge Fig. 8.13 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8

Processing adds a tail to the 3' end of eukaryotic mRNAs Fig. 8.14 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8

RNA splicing removes introns Exons – sequences found in a gene’s DNA and mature mRNA (expressed regions) Introns – sequences found in DNA but not in mRNA (intervening regions) Some eukaryotic genes have many introns Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8

The human dystrophin gene: An extreme example of RNA splicing Splicing removes introns from a primary transcript Fig. 8.15 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8

RNA processing splices out introns and joins adjacent exons Short sequences in the primary transcript dictate where splicing occurs Fig. 8.16a Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8

RNA processing splices out introns and joins adjacent exons (cont) Two sequential cuts remove an intron Fig. 8.16b Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8

Splicing is catalyzed by the spliceosome Fig. 8.17 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8

Alternative splicing can produce two different mRNAs from the same gene Fig. 8.18a Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8

Trans-splicing combines exons from different genes Occurs in C. elegans and a few other organisms Fig. 8.18b Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8

Translation: From mRNA to protein Transfer RNAs (tRNAs) mediate translation of mRNA codons to amino acids Translation takes place on ribosomes that coordinate movement of tRNAs carrying specific amino acids tRNAs are short single-stranded RNAs of 74 – 95 nt Each tRNA has an anticodon that is complementary to an mRNA codon A specific tRNA is covalently coupled to a specific amino acid (charged tRNA) Base pairing between an mRNA codon and an anticodon of a charged tRNA directs amino acid incorporation into a growing polypeptide Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8

Some tRNAs contain modified bases Fig. 8.19a Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8

Three levels of tRNA structure Nucleotide sequence is the primary structure Secondary structure (cloverleaf shape) is formed because of short complementary sequences within the tRNA Tertiary structure (L shape) is formed by 3-dimensional folding Fig. 8.19b Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8

Aminoacyl-tRNA synthetases catalyze attachment of amino acids to specific tRNAs Each aminoacyl-tRNA synthetase recognizes a specific amino acid and the structural features of its corresponding tRNA Fig. 8.20 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8

Base pairing between an mRNA codon and a tRNA anticodon determines which amino acid is added to a growing polypeptide Fig. 8.21 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8

Wobble: Some tRNAs recognize more than one codon for the amino acid they carry Fig. 8.22 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8

A ribosome has two subunits composed of RNA and protein Fig. 8.23a Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8

Mechanism of translation Initiation stage - start codon is AUG at 5’ end of mRNA In bacteria, initiator tRNA has formylated methionine (fMet) Elongation stage - amino acids are added to growing polypeptide Ribosomes move in 5’-to-3’ direction along mRNA 2-15 amino acids added to C terminus per second Termination stage - polypeptide synthesis stops at the 3' end of the reading frame Recognition of nonsense codons Polypeptide synthesis halted by release factors Release of ribosomes, polypeptide, and mRNA Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8

Different parts of a ribosome have different functions Small subunit binds to mRNA Large subunit has peptidyl transferase activity Three distinct tRNA binding areas – E, P, and A sites Fig. 8.23b Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8

Translation of mRNAs on ribosomes: Initiation phase in prokaryotes Ribosome binding site consists of a Shine-Dalgarno sequence and an AUG Three sequential steps: small ribosomal subunit binds first, fMet-tRNA positioned in P site, large subunit binds Initiation factors (not shown) play a transient role Fig. 8.25 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8

Translation of mRNAs on ribosomes: Initiation phase in eukaryotes Small ribosomal subunit binds to 5' cap, then scans the mRNA for the first AUG codon Initiator tRNA carries Met (not fMet) Fig. 8.25 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8

Translation of mRNAs on ribosomes: Elongation phase Addition of amino acids to C-terminus of polypeptide Charged tRNAs ushered into A site by elongation factors (not shown) Fig. 8.25 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8

Polyribosomes consist of several ribosomes translating the same mRNA Simultaneous synthesis of many copies of a polypeptide from a single mRNA Fig. 8.25 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8

Translation of mRNAs on ribosomes: Termination phase No normal tRNAs carry anticodons for the stop codons Release factors bind to the stop codons Release of ribosomal subunits, mRNA, and polypeptide Fig. 8.25 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8

Posttranslational processing can modify polypeptide structure (a) Cleavage may remove an amino acid (c) Chemical constituent addition may modify a protein (b) Cleavage may split a polyprotein Fig. 8.26 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8

Differences between prokaryotes and eukaryotes in gene expression Overview Table 8.1 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8

Differences between prokaryotes and eukaryotes in gene expression (cont) Transcription Table 8.1 (cont) Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8

Differences between prokaryotes and eukaryotes in gene expression (cont) Translation Table 8.1 (cont) Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8

A computerized analysis of gene expression in C A computerized analysis of gene expression in C. elegans: A comprehensive example C. elegans is an ~ 1 mm roundworm that lives in soil Ideal organism for genetic analysis Small size, short life cycle, prolific reproduction C. elegans genome contains roughly 20,000 genes 15% encode components of gene expression machinery 60 genes encode parts of ribosomes 300 genes encode transcription factors 695 tRNA genes, 100 rRNA genes, 72 snRNA genes Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8

Landmarks in the collagen gene of C. elegans Nucleotide sequences of genomic DNA and all mRNA can be obtained for many genes (described in Chapters 9 and 10) Allows detailed analysis of gene structure based on computational analysis of sequences Fig. 8.27a Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8

Sequence of a C. elegans collagen gene, mRNA, and polypeptide Fig. 8.27b Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8

Types of mutations in the coding sequence of genes Fig. 8.28a Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8

Mutations in the coding sequence of a gene can alter the gene product Missense mutations replace one amino acid with another Conservative – chemical properties of mutant amino acid are similar to the original amino acid e.g. aspartic acid [(-)charged]  glutamic acid [(-)charged] Nonconservative – chemical properties of mutant amino acid are different from original amino acid e.g. aspartic acid [(-)charged]  alanine (uncharged) Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8

Mutations in the coding sequence of a gene can alter the gene product (cont) Nonsense mutations change codon that encodes an amino acid to a stop codon (UGA, UAG, or UAA) Frameshift mutations result from insertion or deletion of nucleotides with the coding region No frameshift if multiples of three are inserted or deleted Silent mutations do not alter the amino acid sequence Degenerate genetic code – most amino acids have >1 codon Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8

Mutations outside the coding sequence can disrupt gene expression Fig. 8.28b Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8

Loss-of-function mutations result in reduced or abolished protein activity Loss-of-function mutations are usually recessive Null (amorphic) mutations – completely block function of a gene product (e.g. deletion of an entire gene) Hypomorphic mutations – gene product has weak, but detectable, activity Fig. 8.29 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8

Loss-of-function mutations result in reduced or abolished protein activity (cont) Some loss-of-function mutations can be dominant Incomplete dominance – phenotype varies with the amount of functional gene product (Fig 8.30) Haploinsufficiency – phenotype is sensitive to gene dosage (i.e. 50% of gene product) (Fig 8.31a) Fig. 8.30 Fig. 8.31a Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8

Loss-of-function mutations result in reduced or abolished protein activity (cont) Some loss-of-function mutations can be dominant-negative Usually occurs in genes that encode multimeric proteins Multimeric protein made of four subunits Kinky allele of fused locus Fig. 8.31c Mutant subunits block the activity of normal subunits Fig. 8.31b Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8

Gain-of-function mutations enhance a function or confer a new activity Gain-of-function mutations are usually dominant Hypermorphic mutations – generate more gene product or the same amount of a more efficient gene product Neomorphic mutations – generate gene product with new function or that is expressed at inappropriate time or place Mutant Wild-type Mutation in Antennapedia gene of Drosophila causes ectopic expression of a leg-determining gene in structures that normally produce antennae Fig. 8.31 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8

Mutations classified by their effects on protein function Table 8.2 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8

The cellular components of gene expression Mutations in genes encoding gene products for transcription, RNA processing, translation, and protein processing are often lethal Some mutations in tRNA genes can suppress mutations in protein- coding genes Table 8.3 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8

A nonsense mutation in a protein-coding gene creates a truncated, nonfunctional protein Fig 8.32a Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8

Nonsense suppression A second, nonsense suppressing mutation in the anticodon of a tRNA gene allows production of a (mutant) full-length polypeptide Fig 8.32b Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 8